Hybridization reactions between surface-bound probes and target molecules in solution may be used to detect the presence of particular biopolymers. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of reacting with target molecules in solution. Such reactions form the basis for many of the methods and devices used in the new field of genomics to probe nucleic acid sequences for novel genes, gene fragments, gene variants and mutations. The ability to clone and synthesize nucleotide sequences has led to the development of a number of techniques for disease diagnosis and genetic analysis. Genetic analysis, including correlation of genotypes and phenotypes, contributes to the information necessary for elucidating metabolic pathways, for understanding biological functions, and for revealing changes in genes which confer disease. New methods of diagnosis of diseases, such as AIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, and the like, rely on the detection of mutations present in certain nucleotide sequences. Many of these techniques generally involve hybridization between a target nucleotide sequence and a complementary probe, offering a convenient and reliable means for the isolation, identification, and analysis of nucleotides.
One typical method involves hybridization with probe nucleotide sequences immobilized in an array on a substrate having a surface area of typically less than a few square centimeters. The substrate may be glass, fused silica, silicon, plastic or other material; preferably, it is a glass slide which has been treated to facilitate attachment of the probes. The mobile phase, containing reactants that react with the attached probes, is placed in contact with the substrate, covered with another slide, and placed in an environmentally controlled chamber such as an incubator. Normally, the reactant targets in the mobile phase diffuse through the liquid to the interface where the complementary probes are immobilized, and a reaction, such as a hybridization reaction, then occurs. Preferably, the mobile phase targets are labeled with a detectable tag, such as a fluorescent tag, or chemiluminescent tag, or radioactive label, so that the reaction can be detected. The location of the signal in the array provides the target identification. The hybridization reaction typically takes place over a time period of up to many hours. During this time, the solution between the glass plates has a tendency to dry out through evaporation along the edges of the slide-slide contact.
Such “biochip” arrays have become an increasingly important tool in the biotechnology industry and related fields. These binding agent arrays, in which a plurality of binding agents are synthesized on or deposited onto a substrate in the form of an array or pattern, find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. Substrate-bound biopolymer arrays, particularly oligonucleotide, DNA and RNA arrays, may be used in screening studies for determination of binding affinity and in diagnostic applications, e.g., to detect the presence of a nucleic acid containing a specific, known oligonucleotide sequence.
The pattern of binding by target molecules to biopolymer probe spots on the biochip forms a pattern on the surface of the biochip and provides desired information about the sample. Hybridization patterns on biochip arrays are typically read by optical means, although other methods may also be used. For example, laser light in the Hewlett-Packard GeneArray Scanner excites fluorescent molecules incorporated into the nucleic acid probes on a biochip, generating a signal only in those spots on the biochip that have a target molecule bound to a probe molecule, thus generating an optical hybridization pattern. This pattern may be digitally scanned for computer analysis. Such patterns can be used to generate data for biological assays such as the identification of drug targets, single-nucleotide polymorphism mapping, monitoring samples from patients to track their response to treatment, and assess the efficacy of new treatments.
Control of the reaction environment and conditions contributes to increased reliability and reproducibility of the hybridization reactions. Reducing the volume of the chamber, and therefore increasing the concentration of reactants, increases the sensitivity of the assay. However, merely placing one slide over another or positioning a cover slip on a slide, as is commonly done, is often insufficient to allow precise control over reaction temperature, duration, mixing, and other reaction parameters. For these reasons, efficient reaction chamber design can improve the results achieved with hybridization techniques.
During hybridization, which is often performed at elevated temperatures, care must be taken that the array does not dry out. Merely placing one slide over another or positioning a cover slip on a slide allows contents to leak or dry out during use, adversely affecting the reaction. In addition, the substrate cannot be tipped from the horizontal without risking that the slide or cover slip will slide off. Maintaining a biochip in a humid environment may reduce drying-out, but offers only an incomplete solution. Secondary containment of the solution, as from applying sealant around the edges of the cover over the array, or enclosing the substrate and cover in a closed assembly, may reduce drying-out but is labor-intensive and time-consuming. In addition, in order to result in optimal hybridization, all parts of the array must be contacted by a liquid with uniformly distributed reactants. If the solution dries out, or is not mixed, different portions of the array will be bathed in different concentrations of reactants, impairing the ability to accurately assess the sample.
It is possible to pre-fabricate the chamber and array before use, and so improve the uniformity of the apparatus, as described, for example, in co-pending, commonly assigned U.S. patent application Ser. No. 09/299,976, filed Apr. 27, 1999, entitled “Adjustable Volume, Sealed Chemical-Solution-Confinement Vessel.” That application describes a chamber formed by bonding a glass substrate into a plastic package. However, such a custom-designed package requires specialized processing equipment, and so cannot be used with arrays produced by a laboratory or by sources of generic arrays.
It is possible to contain fluids and reduce drying out in a hybridization or other reaction chamber by providing an O-ring or gasket material between the substrate and cover. However, typical O-rings are about 1.5 to 1.8 mm thick. Using O-rings for sealing would thus leave a relatively large space between the substrate and cover, and would thus result in a reaction chamber requiring a large a volume of reactants. In addition, the O-ring or gasket material would be exposed to the reactants and buffers and may have a deleterious effect on the assay through leaching of contaminants into the reaction chamber and through removal of target molecules out of the reaction chamber by non-specific binding.
Inadequate mixing is a particular problem in chemical and biological assays where very small samples of chemical, biochemical, or biological fluids are typically involved. Inhomogeneous solutions resulting from inadequate mixing can lead to poor hybridization kinetics, low efficiency, low sensitivity, and low yield. With inadequate mixing, diffusion becomes the only means of transporting the reactants in the mobile phase to the interface or surface containing the immobilized reactants. In such a case, the mobile phase can become depleted of reactants near the substrate as mobile molecules become bound to the immobile phase. Also, if the cover is not exactly parallel to the plane of the substrate, the height of the fluid film above the probe array will vary across the array. Since the concentration of target molecules will initially be constant throughout the solution, there will be more target molecules in regions where the film is thicker than in regions where it is thinner, leading to artifactual gradients in the hybridization signal.
Thus, problems associated with hybridization under a cover include drying out of the sample (unless the solution is carefully contained and the humidity of the environment precisely controlled), the need for secondary containment, the inability to ensure that the fluid thickness is uniform across the array, and the inability to mix the solution during hybridization.
Methods for mixing relatively large volumes of fluids usually utilize conventional mixing devices that mix the fluids by shaking the container, by a rapid mechanical up and down motion, or by the use of a rocking motion that tilts the container filled with the fluids in a back and forth motion. The conventional mixing methods normally cannot be utilized for thin films of fluid because the capillary strength of the containment system often significantly exceeds the forces generated by shaking or rocking, thereby preventing or minimizing fluid motion in the film. This is because most or all of the fluid is so close to the walls of the chamber that there is virtually no bulk phase, so that surface interactions predominate.
In some instances, bubbles have been used to help mix large volumes of liquids. For example, U.S. Pat. No. 5,443,985 to Lu et al. and U.S. Pat. No. 5,605,653 to DeVos describe the mixing and aeration of large volumes of liquid, such as a culture medium in a cell culture bioreactor by introducing extraneous gas at the bottom of the reactor thereby creating bubbles that travel upwards, thus mixing the liquid medium. In another context, U.S. Pat. No. 5,275,787 to Yaguchi et al. describes the use of thermal energy to generate a bubble that is then used to discharge a sample liquid containing individual particles. The generation of the bubble and its use as an optical switching element for devices that have uses in telecommunication systems and data communication systems is described in U.S. Pat. No. 5,699,462 to Fouquet et al. and U.S. Pat. No. 4,988,157 to Jackel et al.
Sample binding to spots on biochip arrays is commonly assessed by optical means, although other methods may also be used. Non-specific optical signals, which may arise due to non-specific binding of targets, irregularities or debris on the substrate, or for other reasons, interferes with the accurate analysis of the sample. High background reduces contrast, making it harder to identify spots bound with target molecules, leading to false negative signals. Spurious spots caused by background effects yield false positives signals, by indicating binding where there is none. Thus, high background signals present problems in the acquisition and analysis of optical signals generated by biochip arrays.
Accordingly, there is a need in the art for an improved device and method for conducting chemical or biochemical reactions on a solid substrate within a thin enclosed chamber, wherein mixing of components is facilitated despite the small volume of the chamber, and further wherein the occurrence of unintended chemical reactions is substantially reduced. It is also desirable that the apparatus and method be such that a sample can be contained for extended times at elevated temperatures without drying out, and without the requirements of secondary containment or humidity control.